J. Entomol. Res. Soc., 22(3): 325-337, 2020 Research Article Print ISSN:1302-0250 Online ISSN:2651-3579

Mitochondrial Genomic Analysis of the Tea Geometrid, Ectropis grisescens Warren, 1894 (: Geometridae) and Its Phylogenetic Implications

Hongling LIU1 Zhiteng CHEN2 Chao LIU3 Xinglong WU4 Kejun XIAO5 Jianhui MAO6 Deqiang PU7*

1,3,4,5,6,7Key Laboratory of Integrated Pest Management of Southwest Crops, Institute of Plant Protection, Sichuan Academy of Agricultural Sciences, Chengdu 610066, CHINA 2School of Grain Science and Technology, Jiangsu University of Science and Technology, Zhenjiang 212004, CHINA e-mails: [email protected], [email protected], [email protected], [email protected], [email protected], [email protected], 7*[email protected] ORCID IDs: 10000-0001-7960-009X, 20000-0002-6331-8978, 30000-0001-8697-5543, 40000-0002-2295-6228, 50000-0001-9911-8380, 60000-0002-0508-6320, 70000-0002-3215-814X

ABSTRACT Ectropis grisescens Warren, 1894 is a major pest species only feeding on tea plants but lacks sufficient molecular data to clarify its genetic characters. Complete mitochondrial genome of the tea geometrid Ectropis grisescens is sequenced for the first time. The mitogenome of E. grisescens is 16,575 bp in length, with an A+T content of 81.2%. The typical set of 37 genes (13 PCGs, 22 tRNA genes and two rRNA genes) are all identified. Most PCGs have standard ATN start codons and TAN stop codons. Most tRNA genes exhibite cloverleaf secondary structures, whereas the dihydrouridine (DHU) arm of trnSer (AGN) is reduced into a small loop. Poly-T stretch, tandem repeats and stem-loop (SL) structures are predicted in the control region, which is long (1550 bp) and highly biased toward A+T nucleotides (92.3%). The mitochondrial phylogenetic analyses using the Bayesian inference (BI) and maximum likelihood methods (ML) consistently recover the generic relationships within Geometridae. E. grisescens is correctly grouped with its congener and the monophyly of Ennominae and Larentiinae are both supported. Key words: Lepidoptera, Geometridae, Ectropis grisescens, Mitochondrial genome.

Liu, H., Chen, Z. Liu, C., Wu, X., Xiao, K., Mao, J., & Pu, D. (2020). Mitochondrial genomic analysis of the tea geometrid, Ectropis grisescens Warren, 1894 (Lepidoptera: Geometridae) and its phylogenetic implications. Journal of the Entomological Research Society, 22(3), 325-337. 326 LIU, H., CHEN, Z. LIU, C., WU, X., XIAO, K., MAO, J., & PU, D. INTRODUCTION Mitochondrial genome (mitogenome) has become one of the most popular molecules used in recent phylogenetic studies. The advantages of using mitogenomic data in phylogenetic studies include low cost, sufficient genetic information for higher taxa, and low rate of rearrangement, etc (Boore, 1999). Meanwhile, the mitochondrial cytochrome oxidase subunit I (cox1) gene is widely used for DNA barcoding in species delimitation and intraspecific genetics (Hebert, Cywinska, Ball, & deWaard, 2003). Structures of mitogenomes are usually conserved with 37 typical set of genes, including13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes and two ribosomal RNA (rRNA) genes (Simon, Buckley, Frati, Stewart, & Beckenbach, 2006). Lepidoptera is the second largest order with about 15,7000 species, and many of them are major economic pests (Stork, 2018; Mullen & Zaspel, 2019). Geometridae is one of the largest families in Lepidoptera, comprising over 21,000 species usually harmful to the agriculture and forestry and about 2000 of which are found in China (Scoble, 1999; Gu & Xin, 2018). Among the considerable large number of geometrids, only 12 species have sequenced mitogenomes available from GenBank. The limited mitogenomes are out of proportion to the large number of species in Geometridae. Ectropis grisescens Warren, 1894 also known as the tea geometrid, is an economically important pest species in Asia and is especially widely distributed in the tropical and subtropical areas of China (Warren, 1894; Jiang et al, 2014). Larvae of E. grisescens prefer to damage the bud and leaves of the tea plants, causing considerable economic loss in the tea industry of China (Ge, Yan & Xiao, 2015). Accumulation of genetic data of E. grisescens is warranted to find new control strategies for this pest from a molecular view. However, the mitogenome of E. grisescens is still undetermined, hindering our understanding of the relationship between E. grisescens and other lepidopteran pests. In this study, the complete mitogenome of E. grisescens is sequenced and described for the first time. The mitogenomic structure, nucleotide compositions, codon usages of PCGs, secondary structures of tRNA genes and the control region are investigated to better understand the genetic characters of this important pest.

MATERIALS AND METHODS

Sampling and DNA extraction Adult specimens of E. grisescens were collected from Wolong Town, Qionglai City, Sichuan Province of China in May of 2019. The specimens were identified by Sichuan Academy of Agricultural Sciences and preserved in 100% ethanol. Total genomic DNA was extracted using the E.Z.N.A.® Tissue DNA Kit (OMEGA, America) and stored at -20 °C until used for mitogenome sequencing. 327 Mitochondrial Genomic Analysis of the Tea Geometrid, Ectropis grisescens Mitogenome sequencing, assembly and annotation After DNA extraction, 1.0 μg of purified DNA was fragmented and used to construct Illumina TruSeq short-insert libraries (insert size = 450 bp) following the manufacturer’s instructions, then sequenced on the Illumina Hiseq 4000 (Shanghai BIOZERON Co., Ltd). Raw reads were filtered prior to assembly. The reads with adaptors, with a low quality score below 20, with over 10% “N” bases, or with very few nucleotides were removed. High-quality reads were used for de novo and reference-guided assembling according to the following steps: 1) Firstly, the filtered reads were assembled into contigs using SOAPdenovo2.04 (Luo et al, 2012); 2) Secondly, the contigs were aligned to the reference mitogenome of suspecta Warren, 1894 (GenBank accession number KY095828) using BLAST; the aligned contigs (≥80% similarity and query coverage) were ordered according to the reference mitogenome; 3) Finally, the clean reads were mapped to the assembled draft mitogenome to calibrate the wrong bases, and the gaps were filled by GapFiller v2.1.1 (https://sourceforge.net/projects/gapfiller/). The complete mitogenome sequence of E. grisescens was deposited into GenBank under the accession number MN792921. Location and secondary structures of the tRNA genes were predicted by MITOS (Bernt et al, 2013). PCGs and rRNA genes were annotated using homology alignments. Open reading frames of PCGs were examined and adjusted by ORF finder (https://www.ncbi.nlm.nih.gov/orffinder/). CGView Server (http://stothard.afns.ualberta.ca/cgview_server/) was used to illustrate the mitogenome of E. grisescens (Grant & Stothard, 2008). Nucleotide composition and codon usage were calculated by MEGA v.6.0 (Tamura, Stecher, Peterson, Filipski, & Kumar, 2013). The two formulas AT-skew = [A–T]/[A+T] and GC-skew = [G–C]/[G+C] were used in the composition skew analysis (Perna & Kocher, 1995). Secondary structures in the control region were predicted by Tandem Repeats Finder (http://tandem.bu.edu/trf/ trf.advanced.submit.html) and DNAMAN v6.0.3.

Phylogenetic analyses PCGs derived from 13 of Geometridae, including E. grisescens sequenced in this study, were used in the phylogenetic analyses (Table 1). Another , Cameraria ohridella Deschka & Dimic 1986 from family Gracillariidae (GenBank accession number KJ508042) was used as the outgroup. Each of the 13 PCGs was respectively aligned by MAFFT, then concatenated into a combined dataset using SequenceMatrix v1.7.8 (Katoh & Standley, 2013). PartitionFinder v2.1.1 was used to determine the optimal nucleotide substitution models and partitioning schemes with the Bayesian Information Criterion (BIC) and a greedy search algorithm (Lanfear, Frandsen, Wright, Senfeld, & Calcott, 2016). Two phylogenetic inferences were conducted, including Bayesian inferences (BI) and Maximum likelihood (ML) analysis. BI analysis was conducted by MrBayes v3.2.6, running 20 million generations, sampling every 1000 generations and executing one cold chain and three hot chains with a burn-in of 25% trees (Ronquist & Huelsenbeck, 2003). All runs of BI analysis were examined by Tracer v.1.5 (Rambaut & Drummond, 2019). ML analysis was performed with RAxML v8, with 1000 bootstrap replicates (Stamatakis, 2014). Both BI and ML trees were depicted with FigTree v1.4.2. 328 LIU, H., CHEN, Z. LIU, C., WU, X., XIAO, K., MAO, J., & PU, D.

Table 1. Species of Lepidoptera used in the phylogenetic study.

Family Species GenBank accession number

Abraxas suspecta KY095828

Apocheima cinerarium KF836545

Biston panterinaria JX406146

Biston perclara KU325536

Biston suppressaria KP278206

Biston thibetaria KJ670146

Geometridae Celenna sp. KM244697

Dysstroma truncata KJ508061

Ectropis grisescens KY095828

Ectropis obliqua KX827002

Jankowskia athleta KR822683

Operophtera brumata KP027400

Phthonandria atrilineata EU569764

Gracillariidae Cameraria ohridella KJ508042

RESULTS AND DISCUSSION

Mitogenome structure and nucleotide composition The complete mitogenome of E. grisescens is a double-strand circular molecule with a length of 16,575 bp, longer than all available mitogenomes of Geometridae. The typical set of 37 genes, including 13 PCGs, 22 tRNA genes and two rRNA genes were all identified in the mitogenome. Among these genes, nine PCGs and 14 tRNA genes are encoded by the majority strand (J-strand); the remaining four PCGs, eight tRNA genes and two rRNA genes are on the minority strand (N-strand) (Fig. 1, Table 2). The mitochondrial gene arrangement of E. grisescens differs from the putative ancestral mitogenome of Drosophila yakuba Burla, 1954 by showing the trnMet-trnIle- trnGln tRNA cluster, which is a very common rearrangement pattern in Lepidoptera (Chen & Du, 2016). The mitogenome of E. grisescens has 41 overlapping nucleotides in seven gene pairs; the longest overlap is found between trnPhe and nad5, which is 17-bp long. There are also 238 intergenic nucleotides (IGNs) scattered in 13 gene gaps, indicating a loose mitogenomic structure of E. grisescens. The whole mitogenome of E. grisescens is biased toward A+T nucleotides (81.2%) with a positive AT-skew and negative GC-skew as commonly found in most other (Table 3) (Wei et al, 2010). The A+T contents are similarly high in the concatenated PCGs, PCGs on J-strand, PCGs on N-strand, tRNA genes and rRNA genes (Table 3). All codon positions of the concatenated PCGs have reversal of strand 329 Mitochondrial Genomic Analysis of the Tea Geometrid, Ectropis grisescens asymmetry (negative AT skew and positive GC-skew), whereas tRNA genes and rRNA genes exhibit a positive AT skew and negative GC-skew. In the 37 mitochondrial genes, the A+T content is highest in trnGlu (92.3%) and lowest in cox1 (72.0%).

Table 2. Mitochondrial genome structure of Ectropis grisescens.

Gene Position (bp) Size (bp) Direction Intergenic nucleotides Anti- or start/stop codons A+T%

trnMet (M) 1–69 69 Forward 0 CAT 73.9

trnIle (I) 70–137 68 Forward 0 GAT 77.9

trnGln (Q) 135–203 69 Reverse –3 TTG 84.1

nad2 268–1269 1002 Forward 64 ATA/TAA 84.0

trnTrp (W) 1276–1344 69 Forward 6 TCA 78.3

trnCys (C) 1337–1404 68 Reverse –8 GCA 83.8

trnTyr (Y) 1405–1471 67 Reverse 0 GTA 79.1

cox1 1479–3009 1531 Forward 7 CGA/T 72.0

trnLeu2 (UUR) 3010–3076 67 Forward 0 TAA 73.1

cox2 3077–3758 682 Forward 0 ATG/T 76.8

trnLys (K) 3759–3830 72 Forward 0 CTT 73.6

trnAsp (D) 3833–3898 66 Forward 2 GTC 87.9

atp8 3899–4066 168 Forward 0 ATT/TAA 91.1

atp6 4060–4737 678 Forward –7 ATG/TAA 79.1

cox3 4741–5529 789 Forward 3 ATG/TAA 72.9

trnGly (G) 5532–5597 66 Forward 2 TCC 90.9

nad3 5595–5951 357 Forward –3 ATA/TAA 82.9

trnAla (A) 5955–6018 64 Forward 3 TGC 82.8

trnArg (R) 6019–6082 64 Forward 0 TCG 76.6

trnAsn (N) 6083–6147 65 Forward 0 GTT 78.5

trnSer1 (AGN) 6148–6213 66 Forward 0 GCT 80.3

trnGlu (E) 6282–6346 65 Forward 68 TTC 92.3

trnPhe (F) 6349–6414 66 Reverse 2 GAA 83.3

nad5 6398–8152 1755 Reverse –17 ATC/TAA 81.3

trnHis (H) 8153–8220 68 Reverse 0 GTG 89.7

nad4 8221–9559 1339 Reverse 0 ATG/T 80.5

nad4l 9559–9849 291 Reverse –1 ATG/TAA 85.6

trnThr (T) 9856–9920 65 Forward 6 TGT 80.0

trnPro (P) 9921–9986 66 Reverse 0 TGG 83.3 330 LIU, H., CHEN, Z. LIU, C., WU, X., XIAO, K., MAO, J., & PU, D.

Table 2. Continued.

Gene Position (bp) Size (bp) Direction Intergenic nucleotides Anti- or start/stop codons A+T%

nad6 9989–10522 534 Forward 2 ATA/TAA 85.6

cytb 10,564–11,715 1152 Forward 41 ATG/TAA 75.6

trnSer2 (UCN) 11,714–11,779 66 Forward –2 TGA 81.8

nad1 11,812–12,750 939 Reverse 32 TTG/TAA 77.2

trnLeu1 (CUN) 12,751–12,819 69 Reverse 0 TAG 82.6

rrnL 12,820–14,194 1375 Reverse 0 – 84.5

trnVal (V) 14,195–14,260 66 Reverse 0 TAC 83.3

rrnS 14,261–15,025 765 Reverse 0 – 85.4

Control region 15,026–16,575 1550 – 0 – 91.9

Fig. 1. Mitochondrial map of Ectropis grisescens. Genes outside the map are transcribed clockwise; genes inside the map are transcribed counterclockwise. GC content and GC skew are shown as inside circles. GC content and GC skew are plotted as the deviation from the average value of the entire mitogenome sequence. 331 Mitochondrial Genomic Analysis of the Tea Geometrid, Ectropis grisescens PCGs, tRNA and rRNA genes No rearrangement or variations were found in the 13 PCGs of E. grisescens. Most PCGs start with the standard ATN codon, including ATA start codon in nad2, nad3 and nad6, ATG in cox2, cox3, cytb, atp6, nad4 and nad4l, ATT in atp8 and ATC in nad5 (Table 2). However, cox1 uses the special start codon CGA, and nad1 uses another special start codon TTG. Ten of the PCGs have the complete stop codon TAA, whereas cox1, cox2 and nad4 contain an incomplete stop codon T, which is common in other lepidopteran species (Garey & Wolstenholme, 1989). The relative synonymous codon usage (RSCU) of the 13 PCGs indicate that TTA (Leu), TCT (Ser) and CGA (Arg) are the most frequently used codons whereas CTG (Leu), CCG (Pro) and AGG (Ser) are the least (Fig. 2). The regular set of 22 tRNA genes are all found in the mitogenome, while the ancestral trnIle-trnGln-trnMet tRNA cluster is rearranged into trnMet-trnIle-trnGln. The 22 tRNA genes have a total length of 1471 bp and an A+T content of 81.6%; each tRNA gene ranges in size from 64 to 72 bp (Table 2). Twenty-one of the tRNA genes can fold into typical cloverleaf secondary structures (Fig. 3), however, the TΨC arms of trnPhe and trnThr are somewhat reduced, and the dihydrouridine (DHU) arm is reduced into a small loop, which is very common in other metazoans (Garey & Wolstenholme, 1989). In these tRNA genes, 12 mismatched base pairs are detected and all of them are G-U pairs. The anticodon (AC) arm of trnSer2 forms two loops, which is uncommon in other lepidopteran species. The anticodons of the 22 tRNA genes of E. grisescens are all identical with other sequenced lepidopterans (Yang, Wei, Hong, Jiang, & Wen, 2009). Two rRNA genes were identified in the conserved locations betweentrnLeu1 and the control region of E. grisescens, with a total length of 2140 bp and an A+T content of 84.8% (Table 3). The large ribosomal RNA (rrnL) gene is 1375-bp long, with an A+T content of 84.5%. The small ribosomal RNA (rrnS) gene is 765-bp in size with an A+T content of 85.4%.

Fig. 2. Relative synonymous codon usage (RSCU) of PCGs in Ectropis grisescens. Full codon families are indicated below the X-axis. 332 LIU, H., CHEN, Z. LIU, C., WU, X., XIAO, K., MAO, J., & PU, D.

Fig. 3. Secondary structures of tRNA genes in the mitogenome of Ectropis grisescens. The tRNA genes are labelled with their corresponding amino acids. Structural elements are illustrated as for trnV. Modified arms are indicated with red arrows. Mismatched pairs are indicated with red circles. 333 Mitochondrial Genomic Analysis of the Tea Geometrid, Ectropis grisescens

Table 3. Nucleotide composition of the mitogenome of Ectropis grisescens.

Regions A% T% G% C% A+T% G+C% AT Skew GC Skew

Whole mitogenome 41.2 40.0 7.7 11.1 81.2 18.8 0.015 –0.181

PCGs 34.4 44.4 11.1 10.1 78.8 21.2 –0.127 0.047

1st codon position 36.3 44.6 9.7 9.4 80.9 19.1 –0.103 0.016

2nd codon position 34.3 44.5 11.9 9.3 78.8 21.2 –0.129 0.123

3rd codon position 32.6 44.1 11.6 11.7 76.7 23.3 –0.150 –0.004

PCGs-J 35.9 41.9 10.1 12.1 77.8 22.2 –0.077 –0.090

1st codon position 37.3 40.6 11.2 10.9 77.9 22.1 –0.042 0.014

2nd codon position 32.6 42.5 11.1 13.8 75.1 24.9 –0.132 –0.108

3rd codon position 37.7 42.5 8.1 11.7 80.2 19.8 –0.060 –0.182

PCGs-N 32.0 48.5 12.5 7.0 80.5 19.5 –0.205 0.282

1st codon position 43.6 39.7 6.7 10.0 83.3 16.7 0.047 –0.198

2nd codon position 38.9 42.8 5.5 12.8 81.7 18.3 –0.048 –0.399

3rd codon position 41.1 45.0 3.3 10.6 86.1 13.9 –0.045 –0.525

tRNA genes 41.6 40.0 8.3 10.1 81.6 18.4 0.020 –0.098

tRNA genes-J 41.9 38.6 10.1 9.4 80.5 19.5 0.041 0.036

tRNA genes-N 41.2 42.5 5.2 11.1 83.7 16.3 –0.016 –0.362

rRNA genes 43.9 40.9 4.9 10.3 84.8 15.2 0.035 –0.355

Control region 41.4 50.5 3.0 5.1 91.9 8.1 –0.099 –0.259

Control region The longest non-coding sequence of the E. grisescens mitogenome is the control region located between rrnS and trnMet as in other lepidopterans (Fig. 1, Table 2). The control region of E. grisescens is 1550-bp in size, longer than all sequenced control regions of Geometridae. The A+T content of the control region is 91.9%, which is very high but still lower than trnGlu (92.3%). As the most variable region in a mitogenome, the control region of E. grisescens contains some secondary structures (Fig. 4): a poly-T leading stretch (15,069-15,088) near the 5’ end; seven complete and a partial tandem repeats (15,095-16,502) with a consensus size of 192 bp; three continuous stem-loop (SL) structures (16510-16575) near the 3′ end of the control region. The large tandem repeat region is the main reason for the large size of the control region in E. grisescens. The tandem repeats and SL structures can act as regulators during the mitogenomic replication and transcription processes (Crozier & Crozier, 1993). However, exact functions of the control regions of Lepidoptera are still unclear. 334 LIU, H., CHEN, Z. LIU, C., WU, X., XIAO, K., MAO, J., & PU, D.

Fig. 4. Predicted structural elements in the control region of Ectropis grisescens. Poly-T stretch is indicated with blue box; tandem repeat units are indicated by orange ellipses; stem-loop structures are depicted showing their nucleotides. Other sequences with high A+T content are shown as red lines.

Phylogenetic analyses The two phylogenetic trees respectively generated by the BI and ML analyses have almost identical topological structures (Figs. 5-6). The overall generic relationships recovered by both phylogenetic inferences are (((((Biston + (Apocheima + Jankowskia)) + Ectropis) + Abraxas) + Celenna) + Phthonandria) + (Dysstroma + Operophtera). In both trees, the two subfamilies Ennominae and Larentiinae are recovered as monophyletic with high support (posterior probability = 1, bootstrap value = 100). The four species of genus Biston are grouped together, but B. panterinaria is clustered with B. thibetaria in the BI tree and with B. perclara in the ML tree. In Ectropis, E. grisescens sequenced herein is correctly supported as the sister group of E. obliqua. The sister group relationships are recovered between genera Apocheima and Jankowskia, and also between Dysstroma and Operophtera. In Ennominae, Phthonandria is supported as the basal group of its subfamily, but this conclusion is only reliable before further mitogenomes of Ennominae are sequenced. The phylogenetic results of this study are generally identical with previous mitochondrial phylogenetic studies (Liu, Xue, Cheng, & Han, 2014; Xu, Chen, Wang, Peng, & Li, 2016; Sun et al, 2017; Li, Wang, Chen, & Han, 2018; Zheng et al, 2018). The mitogenomic data has been widely used in Lepidoptera during recent years and considerable efforts have been spent on the molecular phylogeny of diverse lepidopterans, however, very few attention was paid for Geometridae, leaving only nine genera with sequenced mitogemones, which is far from inferring a robust mitochondrial phylogeny of Geometridae. Additional sampling and mitogenome sequencing of each geometrid genus are vital works to clarify the phylogeny and evolution of this biodiverse and economically important insect group. 335 Mitochondrial Genomic Analysis of the Tea Geometrid, Ectropis grisescens

Fig. 5. Phylogenetic relationships within Geometridae inferred by Bayesian inference. Numbers at the nodes are posterior probabilities. The subfamily names are listed after the species. The tree was rooted with the outgroup Cameraria ohridella.

Fig. 6. Phylogenetic relationships within Geometridae inferred by maximum likelihood analysis. Numbers at the nodes are bootstrap values. Subfamily names are shown after the species. The tree was rooted with the outgroup Cameraria ohridella.

ACKNOWLEDGEMENTS This work was supported by the Frontier Discipline Fund of Sichuan Academy of Agricultural Sciences (2019QYXK032), National Key R & D Program of China (2016YFD0200900) and Sichuan Tea Innovation Team of National Modern Agricultural Industry Technology System [sccxtd-2020-10]. 336 LIU, H., CHEN, Z. LIU, C., WU, X., XIAO, K., MAO, J., & PU, D. REFERENCES

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Received: July 21, 2020 Accepted: November 14, 2020